Abstract Rationale Pulmonary disease is a major cause of morbidity and mortality in children with Down syndrome (trisomy 21; T21). We previously identified structural and molecular alterations in human prenatal T21 lungs accompanied by heightened type I interferon activity (IFN-I). Although often associated with immune response, IFN-I has also been implicated in regulation of cellular differentiation and proliferation. We hypothesized that IFN-I perturbs T21 lung development in a tissue-dependent manner. Methods IFN-I components were localized in prenatal T21 and age- and sex-matched non-T21 lungs using fluorescent in situ hybridization (FISH). Non-T21 lung explants were cultured for 72h with or without 200ng IFNβ and analyzed by transcriptomics, while T21 explants were treated with the JAK inhibitor Ruxolitinib. Epithelial buds and fibroblasts from T21 and non-T21 lungs were isolated and either co-cultured in various combinations for 5days or exposed to IFNβ or Ruxolitinib as indicated. Fibroblast-secreted IFNβ was quantified by ELISA in conditioned media collected from 72h fibroblast cultures. All cultures were subsequently assessed by immunofluorescence (IF) and gene expression analyses. Results FISH demonstrated elevated expression of IFNAR1, IFNAR2 and IFNβ in T21 lungs, enriched in mesenchyme (p 0.05). IFNβ treatment of non-T21 explants increased epithelial airway dilatation (p = 0.0144) and decreased proliferation (p 0.01). Transcriptomics confirmed IFN-I upregulation in both epithelium and mesenchyme, with greater induction in mesenchyme. Epithelial progenitor balance shifted, with reduced SOX2 (p = 0.0003) and TP63 (p = 0.0099) and increased SOX9 (p = 0.02), alongside higher expression of alveolar associated markers SFTPC and SFTPB (p 0.05). Pathway analysis confirmed overexpression of surfactant-associated pathway in IFNβ treated explants compared to controls. Alternatively, Ruxolitinib-treated T21 explants demonstrated diminished airway dilatation, restored proliferation, increased TP63, and decreased SOX9 and alveolar markers (gene and protein; p 0.05). Although cell-cell communication (CellChat) showed similar interaction counts between the epithelium and mesenchyme of untreated explants compared to IFNβ-treated, the interaction strength in IFNβ-treated was greater (2.4 vs 1.4), with altered signaling networks linked to development and distalization. Furthermore, signals from mesenchyme to epithelium predominated. IFNβ ELISA on fibroblast conditioned media confirmed significantly higher release from T21 fibroblasts compared to non-T21 (p = 0.0002). In co-cultures, T21 fibroblasts, regardless of epithelial genotype, decreased TP63 and increased SFTPC, recapitulating IFNβ exposure. Conclusions Our results show that excess IFN-I signaling, primarily from mesenchyme, directs epithelial differentiation and suppresses proliferation, driving structural and cellular abnormalities comparable to what is observed in prenatal T21 lungs. Ruxolitinib mitigates these effects, designating IFN-I signaling as a tractable target in T21 lung disease. This abstract is funded by: NIH/NHLBI
Belgacemi et al. (Fri,) studied this question.